Aetiology

Prion diseases of both humans and animals are associated with the accumulation in the brain of an abnormal, partially protease-resistant, isoform of a host-encoded glycoprotein known as prion protein (PrP). The disease-related isoform, PrP Sc, is derived from its normal cellular precursor, PrP C, by a post-translational process that involves a conformational change. PrPC is rich in a-helical structure while PrP Sc appears to be predominantly composed of b-sheet structure. According to the 'protein-only' hypothesis,(7) an abnormal PrP isoform(8) is the principal, and possibly the sole, constituent of the transmissible agent or prion. PrP Sc is hypothesized to act as a conformational template, promoting the conversion of PrPC to further PrPSc. PrPC appears to be poised between two radically different folding states, and a and b forms of PrP can be interconverted in suitable conditions.(9) Soluble b-PrP aggregates in physiological salt concentrations to form fibrils with morphological and biochemical characteristics closely similar to PrP Sc. A molecular mechanism for prion propagation can now be proposed/9) Prion replication, with recruitment of PrPC into the aggregated PrPSc isoform, may be initiated by a pathogenic mutation (resulting in a PrP C predisposed to form b-PrP) in inherited prion diseases, by exposure to a 'seed' of PrPSc in acquired cases, or as a result of the spontaneous conversion of PrP C to b-PrP (and subsequent formation of aggregated material) as a rare stochastic event in sporadic prion disease.

The human PrP gene (PRNP) is a single-copy gene located on the short arm of chromosome 20 and was an obvious candidate for genetic linkage studies in the familial forms of CJD and GSS, which both showed an autosomal dominant pattern of disease segregation. A turning point in understanding the human prion diseases was the identification of mutations in the prion protein gene in familial CJD and GSS in 1989. The first mutation to be identified in PRNP was in a family with CJD and constituted a 144-bp insertion into the coding sequence. (1„9 A second mutation was reported in two families with GSS and genetic linkage was confirmed between this missense variant at codon 102 and GSS, confirming that GSS was an autosomal dominant Mendelian disorder. (!D Uniquely, these diseases are therefore both inherited and transmissible. Current evidence suggests that around 15 per cent of prion diseases are inherited and at least 20 coding mutations in PRNP are now recognized.(1.2)

With the exception of the rare iatrogenic CJD cases mentioned above, most prion disease occurs as sporadic CJD. While, by definition, there will not be a family history in sporadic cases, mutations are seen in occasional apparently sporadic cases, as with a late-onset disease the family history may not be apparent or non-paternity may occur. However, in the majority of sporadic CJD cases there is neither a coding mutation nor a history of iatrogenic exposure. Human prion diseases can therefore be subdivided into inherited, sporadic, and acquired forms. However, a common PrP polymorphism at residue 129, where either methionine or valine can be encoded, is a key determinant of genetic susceptibility to acquired and sporadic prion diseases, the large majority of which occur in homozygous individuals/,1. 14) This protective effect of PRNP codon 129 heterozygosity is also seen in some of the inherited prion diseases. (1. ,,!6)

The aetiology of sporadic CJD remains unclear. It has been speculated that these cases might arise from somatic mutation of PRNP or spontaneous conversion of PrPC to PrPSc as a rare stochastic event. The alternative hypothesis, that such cases arise as a result of exposure to an environmental source of either human or animal prions, is not supported by epidemiological evidence. (1Z>

A major problem for the 'protein-only' hypothesis of prion propagation has been how to explain the existence of multiple isolates or strains of prions which have distinct biological properties. Understanding how a protein-only infectious agent could encode such phenotypic information has been of considerable biological interest. However, it is now clear that prion strains can be distinguished by differences in the biochemical properties of PrP Sc. Prion strain diversity appears to encoded by differences in PrP conformation and pattern of glycosylation. (1.9 A molecular strain typing approach based on these characteristics has allowed the identification of four main types amongst CJD cases, sporadic and iatrogenic CJD being of PrP Sc types 1-3, while all vCJD cases are associated with a distinctive type 4 PrPSc type/.1,19) A similar PrPSc type to that seen in vCJD is seen in BSE and BSE when transmitted to several other species. (18> Such molecular strain typing strongly supported the hypothesis that vCJD was human BSE. This conclusion was strengthened by subsequent transmission studies of vCJD into both transgenic and conventional mice which argued that cattle BSE and vCJD were caused by the same strain.(2 ,2D Such studies are allowing a molecular classification of human prion diseases; it is likely that additional PrP Sc types or strains will be identified. This may well open new avenues of epidemiological investigation and offer insights into causes of 'sporadic' CJD. The ability of a protein to encode a disease phenotype has important implications in biology, as it represents a non-Mendelian form of transmission. It would be surprising if this mechanism had not been used more widely during evolution such that prion biology may prove to be of far wider relevance.

Transmission of prion diseases between different mammalian species is limited by a so-called 'species barrier'. (22) Early studies of the molecular basis of the species barrier argued that it principally resided in differences in PrP primary structure between the species from which the inoculum was derived and the inoculated host. Transgenic mice expressing hamster PrP were, unlike wild-type mice, highly susceptible to infection with hamster prions. (23) That most sporadic and acquired CJD occurred in individuals homozygous at PRNP polymorphic codon 129 supported the view that prion propagation proceeded most efficiently when the interacting PrP Sc and PrPC were of identical primary structure/1,14) However, it has been long recognized that prion strain type affects ease of transmission to another species. Interestingly, with BSE prions the strain component to the barrier seems to predominate, with BSE not only transmitting efficiently to a range of species, but maintaining its transmission characteristics even when passaged through an intermediate species with a distinct PrP gene. (2 25) The term 'species-strain barrier' or simply 'transmission barrier' may be preferable.(25) Both PrP amino acid sequence and strain type affect the three-dimensional structure of glycosylated PrP which will presumably, in turn, affect the efficiency of the protein- protein interactions thought to determine prion propagation. Contribution of other components to the species barrier are possible and may involve interacting cofactors which mediate the efficiency of prion propagation, although no such factors have yet been identified.

The species barrier between cattle BSE and humans cannot be directly measured but can be modelled in transgenic mice expressing human PrP C, which produce human PrPSc when challenged with human prions.(26) When such mice, expressing both human PrP valine 129 (at high levels) and mouse PrP, are challenged with BSE, three possibilities could be envisaged: these mice could produce human prions, murine prions, or both. In fact, only mouse prion replication could be detected. Although there are caveats with respect to this model, particularly that human prion propagation in mouse cells may be less efficient that that of mouse prions, this result would be consistent with the bovine to human barrier being higher than the bovine to mouse barrier for this PRNP genotype. In the second phase of these experiments, mice expressing only human PrP were challenged with BSE. While CJD isolates transmit efficiently to such mice at around 200 days, only infrequent transmissions at over 500 days were seen with BSE, consistent with a substantial species barrier for this human PRNP genotype.(27) However, it is important to repeat these studies in mice expressing only human PrP methionine 129 and in heterozygotes. So far, BSE appears to have transmitted only to humans of PRNP codon 129 methionine homozygous genotype.

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